Project Summary The US opioid epidemic continues to escalate at a rapid pace. Synthetic opioids, primarily illicitly manufactured fentanyl and its analogues (IMFA), became the leading cause of US drug overdose deaths in 2016. The current epidemic is characterized by a rapidly evolving drug supply often tainted with IMFA, which are highly potent and deadly if used unknowingly. Accurate and easy-to-use methods?such as fentanyl test strips (FTS)?that can allow people who use drugs to detect the presence of these chemicals in the illicit drug supply prior to use could help to increase awareness of IMFA and promote safety behaviors to reduce overdose risk. We propose the first study to validate an immunoassay-based FTS to assess its performance in qualitatively detecting the presence of IMFA. This unique study will leverage existing infrastructure and collaborations across criminal justice, biomedical research, and public health. The study aims are to: 1) Compare the limit of detection (LOD) of FTS to high-precision Liquid Chromatography with Tandem Mass Spectrometry (LC- MS/MS) using serial dilution of fentanyl and ?7 fentanyl analogue standards; and 2) Examine the sensitivity and specificity of FTS compared to LC-MS/MS in detecting IMFA in illicit drugs (e.g., synthetic psychoactive cannabinoids, pills, cocaine: N=345 total) collected by the Baltimore Police Department (BPD). To achieve Aim 1, we will conduct a serial analysis of fortified samples of fentanyl and high-priority fentanyl analogues, as well as known blank samples using FTS and LC-MS/MS. The LOD is the lowest concentration of an analyte that consistently yields signal greater than the average signal of the blank. We will also test the repeatability of readings, and assess the impact of background drug interference (e.g., heroin, cocaine, methamphetamine, MDMA) on LOD readings. To achieve Aim 2, we will compare the sensitivity and specificity of FTS against gold standard LC-MS/MS in detecting IMFA in illicit drugs, by testing n=225 IMFA-positive and n=120 IMFA- negative drug samples (N=345 total). We will also conduct quantitative testing on all samples using High Performance Liquid Chromatography (HPLC) to explore whether the detection ability of the FTS is impacted by relative IMFA concentration. All testing will be conducted at the Johns Hopkins Advanced Clinical Chemistry Diagnostics Laboratory (ACCDL) in Baltimore, Maryland in partnership with BPD. This proposal is directly responsive to current NIH priorities on developing innovative analytic detection methods to identify and intervene on dangerous synthetic psychoactive drugs. FTS could be a potentially powerful tool in mitigating the impact of IMFA in the evolving illicit drug supply and therefore their validation would be a valuable addition to our toolkit in meeting this pressing public health challenge.